1. Immunologic Laboratory Tests Kristine Krafts, M.D. | September 17, 2013

2. Immunologic Lab Tests Outline • Agglutination reactions • DAT • IAT • Immunofluorescence • ELISA • Western blot • Flow cytometry

3. Things to Remember About Each Test • What does it measure? • Where does the Ag-Ab interaction occur? • How is the Ag-Ab complex detected?

4. Immunologic Lab Tests Outline • Agglutination reactions

5. Agglutination Reactions: Purpose • Detection of Ag or Ab in patient specimen • Examples: • blood typing • testing for antibodies to infectious agents • testing for Hemophilusinfluenzae type B capsular antigen in CSF

6. Agglutination Reactions: Method • Use particles coated with Ag or Ab • Add patient’s serum (containing Ab or Ag) • See if particles clump

9. Agglutination Reactions: Interpretation Clumping = patient has the antibody (or antigen)

10. negative positive

11. Immunologic Lab Tests Outline • Agglutination reactions • DAT

12. DAT: Purpose • Detection of Ab (or C’) on patient’s red cells • Also called the direct Coombs Test • Performed in patients with hemolytic anemia

13. DAT: Method • Use patient’s red cells (coated with Ab) • Add anti-human globulin (AHG) (Coombs reagent) • Look for agglutination

14. patient red cells + AHG = agglutination

15. DAT: Interpretation Clumping = patient red cells are coated with antibody and/or complement

16. Immunologic Lab Tests Outline • Agglutination reactions • DAT • IAT

17. IAT: Purpose • Detection of antibodies to red cell antigens • Also called the indirect Coombs Test • Performed as part of pre-transfusion testing • antibody screen • cross-match

18. IAT: Method • Use patient serum (containing Ab) • Add donor RBCs (coated with Ag) • Add anti-human globulin (Coombs reagent) • Look for agglutination

19. patient Ab donor RBC Ab-coated donor RBC + = + AHG agglutination =

20. ANTIBODY SCREENING patient serum (without red cell Ab) patient serum (with red cell Ab) reagent RBC (with red cell Ag) reagent RBC (with red cell Ag) AHG AHG no agglutination (negative test) agglutination (positive test)

21. IAT: Interpretation Clumping = patient has an antibody to the donor (or reagent) red cells

22. Immunologic Lab Tests Outline • Agglutination reactions • DAT • IAT • Immunofluorescence

23. Immunofluorescence: Purpose • Detection of a specific antigen or antibody in a specimen • Examples: • detection of bacterial organisms • detection of antigen-antibody complexes

24. Immunofluorescence: Methods • Fix specimen on slide • Add antibodyspecific for the desired antigen • Look for fluorescence Direct Indirect • Fix specimen on slide • Add antibody specific for the desired antigen • Add second antibody • Look for fluorescence

30. Immunofluorescence: Interpretation Fluorescence = patient has the antigen

31. Immunologic Lab Tests Outline • Agglutination reactions • DAT • IAT • Immunofluorescence • ELISA

32. ELISA: Purpose • Detection of antibodies in patient specimen • Examples: • home pregnancy tests • HIV tests • tests for some coagulation factors, cytokines, and autoantibodies

33. ELISA: Method • Add patient specimen to well coated with ligand • Add AHG with enzyme attached • Add substrate • Measure color change

35. ELISA: Interpretation Color change = patient has the antibody

36. ELISA: Variations Sandwich immunoassay • detects antigen (not antibody) • coat well with antibody • rest is like ELISA Radioimmunoassay • detects antibody or antigen • detector is a radioactive substance • otherwise like ELISA or sandwich immunoassay

38. Immunologic Lab Tests Outline • Agglutination reactions • DAT • IAT • Immunofluorescence • ELISA • Western blot

39. Western Blot: Purpose • Detection of antibodies in patient specimen • Most common example: HIV test

40. Western Blot: Method • Make a protein suspension of the target of the antibody you’re looking for (e.g., HIV) • Electrophorese the suspension onto a little gel strip • Apply the patient’s specimen (containing antibodies) to the strip • Add AHG that has an enzyme attached • Add substrate and look for bands

43. Western Blot: Interpretation Bands on strip = patient has antibodies to corresponding proteins

44. Enough bands = patient is “positive”

45. Immunologic Lab Tests Outline • Agglutination reactions • DAT • IAT • Immunofluorescence • ELISA • Western blot • Flow cytometry

46. Flow Cytometry: Purpose • Characterization of cell size, complexity, antigens • Examples: • diagnosis of leukemia and lymphoma • determination of CD4/CD8 counts in patients with HIV